Endogenous biosynthesis of docosahexaenoic acid(DHA)regulates fish oocyte maturation by promoting pregnenolone production

Omega-3 polyunsaturated fatty acids(n-3 PUFAs),particularly docosahexaenoic acid(22:6n-3,DHA),play crucial roles in the reproductive health of vertebrates,including humans.Nevertheless,the underlying mechanism related to this phenomenon remains largely unknown.In this study,we employed two zebrafish genetic models,i.e.,elovl2^(-/-)mutant as an endogenous DHAdeficient model and fat1(omega-3 desaturase encoding gene)transgenic zebrafish as an endogenous DHA-rich model,to investigate the effects of DHA on oocyte maturation and quality.Results show that the elovl2^(-/-)mutants had much lower fecundity and poorer oocyte quality than the wild-type controls,while the fat1 zebrafish had higher fecundity and better oocyte quality than wildtype controls.DHA deficiency in elovl2^(-/-)embryos led to defects in egg activation,poor microtubule stability,and reduced pregnenolone levels.Further study revealed that DHA promoted pregnenolone synthesis by enhancing transcription of cyp11a1,which encodes the cholesterol side-chain cleavage enzyme,thereby stabilizing microtubule assembly during oogenesis.In turn,the hypothalamic-pituitary-gonadal axis was enhanced by DHA.In conclusion,using two unique genetic models,our findings demonstrate that endogenously synthesized DHA promotes oocyte maturation and quality by promoting pregnenolone production via transcriptional regulation of cyp11a1.

A novel ubiquitin carboxyl terminal hydroase is involved in toad oocyte maturation

p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13suc1-agaroseaffinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accessionnumber: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functionaldomains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit theprogesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant proteinp28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminai hydrolases (UCHs). The results in this paperreveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in theprocess of progesterone-induced oocyte maturation possibly through an involvement in protein turnover anddegradation.

Resveratrol compares with melatonin in improving in vitro porcine oocyte maturation under heat stress

Background： Resveratrol, an important phyto-antioxidant commonly found in grapes, mulberry, and other plants,has a variety of functions including anti-aging, anti-cancer and anti-inflammatory activities. In the current study, we investigated the beneficial effects of resveratrol on in vitro porcine oocyte maturation under heat stress（HS）. The effect of resveratrol, melatonin and their combination on alleviating HS was compared according to the maturation rate of oocytes and the development competence of embryos after parthenogenetic activation（PA）.Results： Supplementation with resveratrol（2.0 μmol/L） not only improved the nuclear maturation but also raised the blastocyst rate of porcine embryos＇ PA from oocytes that underwent HS by increasing their glutathione（GSH）level, reducing reactive oxygen species（ROS） and up-regulating the expression of Sirtuin 1（SIRT1）. It was also found that melatonin（10^-7mol/L） and the combination of resveratrol（2.0 μmol/L） plus melatonin（10^-7mol/L） exhibited more potent effects than resveratrol alone regarding their protective activities on oocyte maturation under HS.Conclusions： This study compared the efficiencies of resveratrol, melatonin and their combination for protecting porcine oocytes from heat stress. The mechanisms are attributed to the fact that each treatment may have different ability to regulate the synthesis of steroid hormones and the expression of mature related genes.

Glyphosate exposure deteriorates oocyte meiotic maturation via induction of organelle dysfunctions in pigs

Background:Recently,defects in mammalian oocytes maturation induced by environmental pollution results in the decreasing animal reproduction.Animal exposed to glyphosate is largely unavoidable because glyphosate is one of the most widely used herbicide worldwide due to its high-efficiency and broad-spectrum effects,which causes glyphosate an environmental contaminant found in soil,water and food.During the last few years,the growing and wider use of glyphosate has raised great concerns about its effects of reproductive toxicity.In this study,using porcine models,we investigated effects of glyphosate on organelle functions during oocyte meiosis.Results:The results showed glyphosate exposure disrupted porcine oocyte maturation.Expression levels of cumulus expansion-related genes were interfered,further indicating the meiotic defects.The damaging effects were mediated by destruction of mitochondrial distribution and functions,which induced ROS accumulation and oxidative stress,also indicated by the decreased mRNA expression of related antioxidant enzyme genes.We also found an interference of endoplasmic reticulum(ER)distribution,disturbance of Ca^(2+)homeostasis,as well as fluctuation of ER stress,showing with the reduced ER stress-related mRNA or protein expression,which could indicate the dysfunction of ER for protein processing and signal transduction in glyphosate-exposed oocytes.Moreover,glyphosate exposure induced the disruption of lysosome function for autophagy,showing with the decrease of LAMP2 expression and autophagy-related genes mRNA expression.Additionally,our data showed the distribution of Golgi apparatus and the functions of ribosome were disturbed after glyphosate exposure,which might affect protein synthesis and transport.Conclusions:Collectively,our study showed that exposed to glyphosate could affect animal reproduction by compromising the quality of oocytes through its wide toxic effects on organelle functions.

In vitro maturation of human oocytes maintaining good development potential for rescue intracytoplasmic sperm injection with fresh sperm

BACKGROUND The outcomes of the use of commercial in vitro maturation(IVM)medium to culture immature oocytes obtained from conventional ovulation induction,followed by rescue intracytoplasmic sperm injection(RICSI),are not ideal.It is thus difficult to widely adopt this approach in clinical practice.Therefore,it is necessary to explore methods for improving the clinical outcome of IVM.AIM To study the effect of sperm on the developmental potential of in vitro-matured oocytes in conventional culture.METHODS This was a retrospective study of patients whose immature oocytes were harvested from conventional oocyte stimulation cycles and underwent ICSI at our hospital between June 2018 and August 2020.RICSI was performed using sperm collected on the day of oocyte harvest(old)and sperm collected on the day of RICSI(fresh)and oocytes matured in vitro after 24 h of culture in conventional medium.The rates of in vitro oocyte maturation,normal fertilization,normal cleavage,day-3 top-quality embryos,and useful blastocyst formation were compared between the two groups.RESULTS In total,102 germinal vesicle(GV)-stage immature oocytes were cultured in the old sperm group.In the fresh sperm group,122 GV-stage immature oocytes were collected and cultured in vitro for 24 h.There were no significant differences in the general conditions of males and females between the two groups(P>0.05).The oocyte maturation,normal fertilization,and normal cleavage rates of the old and fresh groups were 51.0%vs 55.7%,61.5%vs 64.7%,and 93.8%vs 93.2%,respectively.None of the rates differed significantly(P>0.05)between the two groups.However,the day-3 top-quality embryo and useful blastocyst rates of the old and fresh sperm groups were 16.6%vs 63.4%;6.67%vs 34.6%,respectively.The day-3 top-quality embryos and useful blastocyst rates of the old sperm group were significantly lower than those of the fresh group(P<0.05).CONCLUSION In vitro maturation with conventional culture medium combined with the use of fresh sperm collected on the day of RICSI is an easy-to-implement strategy for patients whose oocytes are completely or mostly immature.

Evaluation of pig oocyte in vitro maturation and fertilization using three gonadotropin-based hormonal compounds

Objective:To evaluate the effect of human chorionic gonadotropins(hCG)and equine chorionic gonadotropins(eCG)on in vitro gilt oocyte maturation and embryonic development,using frozen semen for fertilization.Methods:Two independent experiments(6 replicates each)were carried out to evaluate gilt oocyte maturation,and fertilization and embryonic development by using ovaries from a local abattoir.Totally,712 oocytes were randomly distributed in four-well dishes to receive Novormon(eCG 5.0 IU),PG600(eCG 5.0 IU and hCG 2.5 IU),Chorulon(hCG 5.0 IU),or no hormones.Oocytes were incubated with 5%CO2,95%air and saturation humidity at 39℃for 44 h.Maturation of the oocytes to metaphaseⅡwas assessed by using the aceto-orcein technique.In addition,741 oocytes were used and randomly distributed in four-well dishes,and then oocyte maturation was carried out as mentioned,but matured oocytes were washed and placed in fertilization medium with frozen-thawed sperm.Gametes were co-incubated for 7 h,and then washed and placed in development medium,and incubated for further 7 days,at which time embryonic development was evaluated.Fertilization and embryo development media were not supplemented with the studied hormones.Results:Novormon(eCG)and PG600(eCG+hCG)treatments significantly improved the percentages of metaphaseⅡoocytes compared to the control group(P<0.05).Furthermore,a significant increase was also observed in the young blastocyst stage between the control group and the PG600 treatment group(P<0.05).Conclusions:Hormonal products Novormon(eCG)and PG600(eCG+hCG)can obtain the highest percentages of in vitro maturation in gilt oocytes;however,this effect is not transferred to fertilization rates.

Kisspeptin stimulates oocyte maturation,and food deprivation modulates the abundance of kisspeptin system in zebrafish gonads

Kisspeptin is a hormone involved in the neuroendocrine control of reproduction in fish.We hypothesized that kisspeptin stimulates oocyte maturation and modulates other reproductive hormones in zebrafish;and the gonadal kisspeptin system in zebrafish is affected during energy unavailability.The main goals of this research were to test in vitro effects of kisspeptin on oocyte maturation and mRNA abundance in zebrafish ovarian follicles and determine how short-term feed restriction affects kisspeptin and its receptors in zebrafish testis and ovary.This study demonstrates the presence of kisspeptin and its receptors in zebrafish(Danio rerio)gonads and its direct action on ovarian follicles in vitro.Kisspeptin(10 ng/mL)induced oocyte maturation,as indicated by germinal vesicle break down at 18-and 24-hours post-treatment.Kisspeptin significantly increased the abundance of mRNAs encoding reproductive hormones and its receptors in zebrafish oocytes.This suggests that kisspeptin-10 affects ovarian functions by modulating other hormones.Reproduction is a process that requires energy.Therefore,whether energy availability affects the kisspeptin system in zebrafish gonads was determined.Food deprivation modulated kisspeptin expression differently in zebrafish testis and ovary.Kiss2 and kiss1ra were upregulated while kiss1rb was downregulated in the testis post-food deprivation.Meanwhile,no changes in kiss in the ovary were found after food deprivation.However,kiss1rb was downregulated in unfed fish at 3-and 7-days post-food deprivation.Overall,our results suggest sex-and tissue-specific changes in the gonadal abundance of the kisspeptin system in zebrafish.The fine tuning of reproduction during energy fluctuations in fish is likely mediated via changes in hormones,including kisspeptin as shown in this research.

Reference gene validation for quantification of gene expression during final oocyte maturation induced by diethylstilbestrol and di-(2-ethylhexyl)-phthalate in common carp

Final oocyte maturation is the key step to successful spawning and fertilization.Quantitative real-time PCR（q PCR） is the technique of election to quantify the abundance of functional genes in such study. Reference gene is essential for correct interpretation of q PCR data. However, an ideal universal reference gene that is stable under all experimental circumstances has not been described. Researchers should validate their reference genes while performing q PCR analysis. The expression of 6 candidate reference genes： 18 s r RNA,28 s r RNA, Cathepsin Z, Elongation factor 1-α, Glyceraldehyde-3-phosphate dehydrogenase andβ-actin were investigated during final oocyte maturation induced by different compounds（DES and DEHP） in common carp（Cyprinus carpio）. Four softwares（Bestkeeper, ge Norm,Norm Finder and Ref Finder） were used to screen the most stable gene in order to evaluate their expression stability. The results revealed that EF1α was highly stable expressed when final oocyte maturation was induced by DES, while gapdh was the most stable gene when final oocyte maturation was induced by DEHP. Stable expressed reference gene selection is critical for all q PCR analysis to get accurate target gene m RNA expression information.

Effects of Ages of Donors and Conditions of Preserving Ovaries on Porcine Oocytes Maturation in vitro and Efficiency of Parthenogenetic Activation

Effects of different ages of donors and different conditions of preserving ovaries onporcine oocytes maturation in vitro and efficiency of parthenogenetic activation werestudied. The experiments included: 1) effects of different temperatures (22, 30, 37, 38.5and 40℃) of preserving ovaries on porcine oocytes maturation in vitro and developmentalpotential; 2) effects of periods of preserving ovaries on porcine oocytes maturation invitro and development in vitro; 3) effects of different ages of donors on porcine oocytesmaturation in vitro and developmental potential. The results of the experiment showed:1) There were no statistical differences (p>0.05) of the parthenogenetic cleavage rate(79.64% vs 76.18%) and blastocyst rate (18.11% vs 33.82%) between oocytes from ovariespreserved at 38.5℃ and those preserved at 37℃. When the preserving temperature wasincreased to 40℃, the cleavage rate (21.68%) and the blastocyst rate (0) were greatsignificantly lower than those at 37℃(p<0.01). The cleavage rate (80.79% vs 76.18%) andblastocyst rate (29.61% vs 33.82%) were not different between 30 and 37℃(p>0.05). Whenthe preserving temperature was decreased to 22℃, the rate of cleavage was not different,but the rate of blastocyst was significantly lower, compared with that at 37℃; 2) Thecleavage and blastocyst rates of the porcine oocytes collected after slaughter 2 or 6hwere not different (p>0.05); 3) The cleavage rate of oocytes from gilts and sows aftermaturation was not different, but the blastocyst rate of the sow group was significantlyhigher than that of gilt group (p<0.05). The blastocyst cell number of sows and giltshowed no difference (p>0.05).

Microbial and metabolomic mechanisms mediating the effects of dietary inulin and cellulose supplementation on porcine oocyte and uterine development

Background:Dietary fiber(DF)is often eschewed in swine diet due to its anti-nutritional effects,but DF is attracting growing attention for its reproductive benefits.The objective of this study was to investigate the effects of DF intake level on oocyte maturation and uterine development,to determine the optimal DF intake for gilts,and gain microbial and metabolomic insight into the underlying mechanisms involved.Methods:Seventy-six Landrace×Yorkshire(LY)crossbred replacement gilts of similar age(92.6±0.6 d;mean±standard deviation[SD])and body weight(BW,33.8±3.9 kg;mean±SD)were randomly allocated to 4 dietary treatment groups(n=19);a basal diet without extra DF intake(DF 1.0),and 3 dietary groups ingesting an extra50%(DF 1.5),75%(DF 1.75),and 100%(DF 2.0)dietary fiber mixture consisting of inulin and cellulose(1:4).Oocyte maturation and uterine development were assessed on 19 d of the 2nd oestrous cycle.Microbial diversity of faecal samples was analysed by high-throughput pyrosequencing(16S r RNA)and blood samples were subjected to untargeted metabolomics.Results:The rates of oocytes showing first polar bodies after in vitro maturation for 44 h and uterine development increased linearly with increasing DF intake;DF 1.75 gilts had a 19.8%faster oocyte maturation rate and a 48.9 cm longer uterus than DF 1.0 gilts(P<0.05).Among the top 10 microbiota components at the phylum level,8increased linearly with increasing DF level,and the relative abundance of 30 of 53 microbiota components at the genus level(>0.1%)increased linearly or quadratically with increasing DF intake.Untargeted metabolic analysis revealed significant changes in serum metabolites that were closely associated with microbiota,including serotonin,a gut-derived signal that stimulates oocyte maturation.Conclusions:The findings provide evidence of the benefits of increased DF intake by supplementing inulin and cellulose on oocyte maturation and uterine development in gilts,and new microbial and metabolomic insight into the mechanisms mediating the effects of DF on reproductive performance of replacement gilts.

Effect of Oocyte Recovery Techniques on in Vitro Production of Swine Embryos

In Vitro production of swine embryos is a valuable tool to generate clones and genetically modified pigs during a short period of time. However, the efficiency of the existing methods is extremely low and the oocyte quality and quantity represent important obstacles on the success of in Vitro production of embryos. Therefore, the aim of this study was to compare the in Vitro maturation, fertilization and subsequent embryo development rates of oocytes recovered by ovary slicing or follicular aspiration. The oocyte recovery rate (grade 1 COC/ovary) was higher (p = 0.0083) in the slicing group when compared to the aspiration group. No differences were observed between groups regarding in Vitro maturation and early cleavage rates. A higher percentage of oocytes recovered by follicular aspiration reached the blastocyst stage after IVF when compared to the ovary slicing method (p = 0.0395). However, no difference on blastocyst cell number was observed. Although the recovery of oocytes using the slicing technique yielded more grade 1 oocytes per ovary than the aspiration method, the number of oocytes that reached the blastocyst stage after IVF by the slicing method was lower when compared with oocytes obtained by aspiration, as observed by lower blastocyst rates. In conclusion, the follicular aspiration is the method of choice for porcine in Vitro embryo production.

Efficacy of Simple Assessment System of Oocyte Maturity in IVF-ET Cycles

Objective To establish and evaluate the efficacy of the simple assessment system of oocyte maturity. Methods A total of 251 couples were enrolled in this study and female patients were divided into two groups. In group Ⅰ, oocytes were inseminated at 6 h after ovum pick-up. In group Ⅱ, oocyte maturity was rapidly categorized by simple assessment system. Mature oocytes were inseminated at 3-4 h after ovum pick-up when oocyte-corona complexes （OCC） exhibited clear thick ring-like halo （RLH） and expanded cumulus cells （CC） or 5-6 h when OCC exhibited RLH and a few clumped and/or dark CC, respectively. Intermediate oocytes were inseminated at 7-8 h when RLH was not found around the OCC and CC were compacted and clumped and/or dark. Results Normal fertilization rate was higher in group Ⅱ （76.5%） than that in group Ⅰ （58.0%） （P〈0.001）. However, abnormal fertilization rate was higher in group Ⅰ（11.3%） than that in group 11 （3.6%） （P〈0.001）. The cleavage （82.6% vs 90.0%）, chemical pregnancy （4. 8% vs 3.9%）, twin pregnancy （6. 7% vs 3.9%） and implantation rate （8.4% vs 10.6%） had no statistically differences between group I and Ⅱ. Rate of clinical and singleton pregnancy was higher in groupⅡ （35.3% and 31.4%） than those in group Ⅰ （24.8% and 18.2%） （P〈0.05）. Conclusion This simple assessment system is useful and effective for evaluation and categorization of the oocyte maturity with better reproductive outcomes.

Current and future therapies for abnormal early embryogenesis with assisted reproductive technology

Each stage of embryonic development,including normal gamete maturation,fertilization,zygotic genome activation,and cleavage,is crucial for human reproduction.Early embryo arrest is a common phenomenon.It is estimated that about 40%–70%of human embryos are arrested at early developmental stages.However,the exact mechanism remains largely uncertain.Embryos can be investigated in vitro by way of the development of in vitro fertilization/intracytoplasmic sperm injection.In addition to iatrogenic factors related to abnormal oocyte/embryo development,multiple gene mutations have been found to be involved in such phenotypes.Based on the knowledge of known etiological factors,several therapies are proposed to improve clinical outcomes.Here,we shed light on current and potential therapies for treating these conditions through reviewing articles and combining with our clinical and research experience.

The effect of antioxidants on increased oocyte Competence in IVM: a review

In vitro maturation(IVM)is considered a potential assisted reproductive technology that is a safer and simpler alternative to conventional in vitro fertilization.It is primarily used in patients with impaired oocyte maturation and for the treatment of infertile women who are at risk of fertility loss.In addition,IVM is currently used mainly in polycystic ovarian syndrome patients with a high ovarian response and is stll considered an experimental option in fertility preservation.Producing highly competent oocytes during IVM is considered a key step in the success of in vitro production(IVP)of embryos.Some factors,such as culture medium conditions and other supplements,have a significant impact on oocyte IVM performance.One of the known disruptors of oocyte developmental competence in IVP is oxidative stress(Os),which is caused by an imbalance between the production and neutralization of reactive oxygen species(ROS).In vitro conditions induce supraphysiological ROS levels due to exposure to an oxidative environment and the isolation of the oocyte from the follicle protective antioxidant milieu.Given the importance of Os in oocyte competence,the establishment of standardized antioxidant IVM systems is critical for improving the overall success of IVP.This review focuses on the main antioxidants tested to protect oocytes against OS in IVM.

Roles of insulin-like growth factor Ⅱ in regulating female reproductive physiology

The number of growth factors involved in female fertility has been extensively studied, but reluctance to add essential growth factors in culture media has limited progress in optimizing embryonic growth and implantation outcomes, a situation that has ultimately led to reduced pregnancy outcomes. Insulin-like growth factor Ⅱ(IGF-Ⅱ) is the most intricately regulated of all known reproduction-related growth factors characterized to date, and is perhaps the predominant growth factor in human ovarian follicles. This review aims to concisely summarize what is known about the role of IGF-Ⅱ in follicular development, oocyte maturation, embryonic development, implantation success, placentation, fetal growth, and in reducing placental cell apoptosis, as well as present strategies that use growth factors in culture systems to improve the developmental potential of oocytes and embryos in different species. Synthesizing the present knowledge about the physiological roles of IGF-Ⅱ in follicular development, oocyte maturation, and early embryonic development should, on the one hand, deepen our overall understanding of the potential beneficial effects of growth factors in female reproduction and on the other hand support development(optimization) of improved outcomes for assisted reproductive technologies.

Acceleration of coasting enhances pregnancy rate in ICSI cycles at risk for ovarian hyperstimulation syndrome

Objective To assess the efficacy of adding Gn-releasing hormone antagonist（GnR HA） on the day of hC G triggering in a long luteal protocol without withholding the agonist in women who are at a risk to develop ovarian hyperstimulation syndrome（OHSS）.Methods This was a retrospective cohort study conducted upon 50 women who have elevated serum estradiol（E2） level 〉4 500 ng/L at the day of ovum triggering with hC G on a long agonist luteal protocol of controlled ovarian stimulation（COS）. When an exaggerated ovarian response was observed around day 10 of stimulation, immediately the next morning at 6 a.m. gonadotropin administration was stopped or reduced, and a single dose of ganirelix acetate（antagonist） was given sc continuation of the agonist dose hC G. Another serum E2 measurement was done at 6 p.m.（after 12 h of antagonist） then hC G, sc 250 mg and choriogonadotropin α were administered 14 h later after antagonist and documented the reduction of E2 level. Oocyte retrieval was conducted after 34-36 h of hC G administration. The measured outcomes were the level of E2 on the day of hC G injection, number of oocytes and their quality, pregnancy rate and the occurrence of OHSS and its grade in case it happened.Results The total dose for recombinant FSH was 25.3±6.4 ampoules（75 IU/ampoule） while it was 11.0±3.0 ampoules for the urinary hM G. A higher oocyte maturation rate（82.8%） and a high fertilization rate（87.8%） were observed. The mean endometrial thickness was 10.1±1.0 mm on the day of hC G triggering. The higher fertilization rate with the good endometrial thickness observed resulting in a higher pregnancy rate（78.0%, 39/50） with statistically significant（P〈0.05）. A significant reduction of E2 level was documented by a percentage around 40% before hC Ginjection. There were no reported cases of severe or moderate OHSS, however 13 cases（26%） were reported to have mild OHSS constituting.Conclusion Acceleration of coasting in cases of OHSS through treatment with GnR HA after pituitary suppression with GnR H agonist（GnR H-a） offered a novel approach to decrease E2 level, avoided cycle cancellation, and maintain excellent oocyte maturation rate, and finally result in high pregnancy rate with prevention of OHSS.

Changes in human sperm motility and DNA fragmentation index after incubation at different temperatures following density gradient centrifugation and swim-up procedures

Objective:The purpose of this study was to evaluate the sperm motility and DNA integrity at different temperatures to analyze whether the sperms are suitable on the second day for insemination of in vitro matured oocytes by intra-cytoplasmic sperm injection(ICSI)following density gradient centrifugation(DGC)and swim-up(SU)procedures.Methods:Semen samples were collected from 30 outpatients who visited the Center for Reproductive Medicine for semen analyses.Following sperm selection by DGC and SU procedures,the liquified semen samples were divided into three groups and incubated at 4,25,and 37°C,respectively.Following incubation for 24,48,and 72 hours,the sperm motility and sperm DNA fragmentation index(DFI)were analyzed.Results:Following the combination of DGC and SU procedures,the sperm motility(91.8%±8.6%vs.50.8%±13.1%)and DFI(5.1%±7.9%vs.13.0%±11.6%)were significantly improved(P<0.01)compared to those without any treatment.The sperm motility of the 3 groups significantly declined(P<0.05)post-incubation compared to that of the groups prior incubation.However,sperm motility significantly increased(76.9%±10.4%)(P<0.05)at 25°C compared to that of the other 2 groups(53.5%±11.0%and 47.6%±10.2%).Sperm DFI significantly increased(P<0.05)at 37°C following incubation for 24 and 72 hours in comparison to that of the other 2 groups.However,the sperm DFI did not significantly increase when the sperm samples were incubated at 4(5.7%±5.9%)and 25°C(6.8%±5.6%)for 24 hours compared to that before incubation(5.1%±7.9%).Conclusions:These results indicate that the sperm quality,in terms of motility and DFI,can be efficiently improved by DGC in combination with SU.Following which,the sperm samples can be incubated at 25°C and be used on the second day for insemination of in vitro matured oocytes by ICSI.